Drug addiction is a major health issue worldwide, and the central focus of the NIDA Center at Rockefeller University. All addictive substances enhance dopamine in the mesolimbic reward circuit from the ventral tegmental area (VTA) to the nucleus accumbens shell (Acb-SH), a limbic brain region included with the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) as components of the extended amygdala. These regions are also targeted by many excitatory inputs, whose physiological actions are largely ascribed to activation of glutamate (NMDA and AMP A) receptors. Glutamatergic transmission is potently modulated by dopamine acting at Dl receptors and corticotrophin releasing factor (CRF) peptides active at CRF type-1 (CRF1) receptors that are prevalent in both the central extended and basolateral (BLA) amygdala. The more cortical-like BLA has bidirectional connections with the VTA-and other limbic structures implicated in emotional behavior and learning of drug/reward associations. Glutamate receptor plasticity and associations with the dopamine and/or CRF systems contribute to persistent drugseeking behaviors that are powerfully influenced by stress. The subcellular changes in receptor distributions occurring in neurons with these identified transmitter phenotypes in individual brain regions are largely unknown. To begin addressing these key questions, Project 3 in the renewal application will combine research strategies using electron microscopic immunolabeling and spatial-temporal deletion (knock-out) of the NR1 NMDA receptor subunit in limbic brain regions critical for drug seeking behaviors. The long-range goal is to test the hypotheses that (1) limbic NMDA receptors have subcellular distributions conducive to regionally selective associations with dopamine and CRF systems, and (2) NR1 gene expression in the VTA and/or BLA is essential for the synaptic targeting and cocaine-induced trafficking of both AMP A and dopamine Dl receptors, and for cocaine conditioned place preference (CPP) influenced by stress. The studies will be conducted in wild-type and NR1 "floxed" (flanked by loxP) mice, some of which will receive acute or chronic (14 day) cocaine given in an escalating "binge" pattern mimicking that seen in human addicts. Project 3 reflects a collaborative effort by investigators in existing projects within the NIDA Center and is totally dependent on the core resources and facilities. The results obtained from Project 3, together with those in other projects in the renewal application, will provide important new information that is essential for understanding and treating drug addiction.